This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The way in which genes are expressed in all of our cells is a highly regulated, complex process involving hundreds of protein factors and several non-coding snRNAs. The process of pre-mRNA splicing is required to remove stretches of RNA called introns, which if not removed would interfere with proper expression of that gene. Almost all human genes contain introns and their faithful and rapid removal is required for proper functioning. The machinery in eukaryotic cells charged with the removal of introns is called the spliceosome, a collection of RNAs and many proteins that rearranges several times during the course of a single intron removal, recruiting factors and ejecting others in a highly coordinated manner. The recent discovery of the penta-snRNP in yeast cells has challenged a paradigm in the mechanism of spliceosome assembly, specifically, that the machinery is largely pre-assembled and that the rearrangements occur within this pre-assembled complex. The single-celled eukaryotic organism Saccharomyces cerevisiae will be used to study the process of spliceosome assembly and the pre-mRNA splicing reaction as it is amenable to genetic and biochemical analysis and executes the pre-mRNA splicing reaction in a way that is chemically identical to the human splicing reaction. This CAREER project will address the mechanism of spliceosome assembly and the means by which the penta-snRNP engages its pre-mRNA substrate. Compositional and functional analyses of the early splicing intermediates will allow a comprehensive biochemical and genetic analysis of the initial events in spliceosome assembly. In conjunction with the Austin Independent School District, this project also endeavors to initiate and execute a comprehensive outreach program involving area high school teachers, administrators and students to provide exposure of interested junior and senior students to modern molecular biology and the educational and research options available to them.